This invention relates to nucleic acids and encoded polypeptides which interact with TGF-xcex2 superfamily receptors and which are negative regulators of signaling by those receptors. The invention also relates to agents which bind the nucleic acids or polypeptides. The invention further relates to methods of using such nucleic acids and polypeptides in the treatment and/or diagnosis of disease.
During mammalian embryogenesis and adult tissue homeostasis transforming growth factor xcex2 (TGF-xcex2) performs pivotal tasks in intercellular communication (Roberts et al., Growth Factors 8:1-9, 1993). The cellular effects of this pleiotropic factor are exerted by ligand-induced hetero-oligomerization of two distantly related type I and type II serine/threonine kinase receptors, Txcex2R-I and Txcex2R-II, respectively (Lin and Lodish, Trends Cell Biol. 11:972-978, 1993; Derynck, Trends Biochem. Sci. 19-:548-553, 1994; Massague and Weis-Garcia, Cancer Surv., 27:41-64, 1996; ten Dijke et al., Curr. Opin. Cell. Biol. 8:139-145, 1996). The two receptors, which both are required for signaling, act in sequence; Txcex2R-I is a substrate for the constitutively active Txcex2R-II kinase (Wrana et al., Nature 370:341-347, 1994; Weiser et al., EMBO J. 14:2199-2208, 1995). TGF-xcex2 forms part of a large family of structurally related proteins which include activins and bone morphogenetic proteins (BMPs) that signal in a similar fashion, each employing distinct complexes of type I and type II serine/threonine kinase receptors (Lin and Lodish, 1993; Derynck, 1994; Massague and Weis-Garcia, 1996; ten Dijke et al., 1996).
Genetic studies of TGF-xcex2-like signalling pathways in Drosophila and Caenorhabditis elegans have led to the identification of mothers against dpp (Mad) (Sekelsky et al., Genetics 139:1347-1358, 1995) and sma (Savage et al., Proc. Nat. Acad. Sci. USA 93:790-794, 1996) genes, respectively. The products of these related genes perform essential functions downstream of TGF-xcex2-like ligands acting via serine/threonine kinase receptors in these organisms (Wiersdorf et al., Development 122:2153-2163, 1996; Newfeld et al., Development 122:2099-2108, 1996; Hoodless et al., Cell 85:489-500, 1996). Vertebrate homologs of Mad and sma have been termed Smads (Derynck et al., Cell 87:173, 1996) or MADR genes (Wrana and Attisano, Trends Genet. 12:493-496, 1996). Genetic alterations in Smad2 and Smad4/DPC4 have been found in specific tumor subsets, and thus Smads may function as tumor suppressor genes (Hahn et al., Science 271:350-353, 1996; Riggins et al., Nature Genet. 13:347-349, 1996; Eppert et al., Cell 86:543-552, 1996). Smad proteins share two regions of high similarity, termed MH1 and MH2 domains, connected with a variable proline-rich sequence (Massague, Cell 85:947-950, 1996; Derynck and Zhang, Curr. Biol. 6:1226-1229, 1996). The C-terminal part of Smad2, when fused to a heterologous DNA-binding domain, was found to have transcriptional activity (Liu et al., Nature 381:620-623, 1996; Meersseman et al., Mech. Dev. 61:127-1400, 1997). The intact Smad2 protein when fused to a DNA-binding domain, was latent, but transcriptional activity was unmasked after stimulation with ligand (Liu et al., 1996).
Different Smads specify different responses using functional assays in Xenopus. Whereas Smad1 induces ventral mesoderm, a BMP-like response, Smad2 induces dorsal mesoderm, an activin/TGF-xcex2-like response (Graff et al., Cell 85:479-487, 1996; Baker and Harland, Genes and Dev. 10:1880-1889, 1996; Thomsen, Development 122:2359-2366, 1996). Upon ligand stimulation Smads become phosphorylated on serine and threonine residues; BMP stimulates Smad1 phosphorylation, whereas TGF-xcex2 induces Smad2 and Smad3 phosphorylation (Hoodless et al., 1996; Liu et al., 1996; Eppert et al., 1996; Lechleider et al., J. Biol. Chem. 271:17617-17620, 1996; Yingling et al., Proc. Nat""l Acad. Sci. USA 93:8940-8944, 1996; Zhang et al., Nature 383:168-172, 1996; Macxc3xadas-Silva et al., Cell 87:1215-1224, 1996; Nakao et al., J. Biol. Chem. 272:2896-2900, 1996). Thus certain Smads are pathway specific. Pathway specific Smads include Smad1, Smad2, Smad3 and Smad5.
Smad4 is a common component of TGF-xcex2, activin and BMP signaling (Lagna et al., Nature 383:832-836, 1996; Zhang et al., Curr. Biol. 7:270-276, 1997; de Winter et al., Oncogene 14:1891-1900, 1997). Smad4 phosphorylation has thus far been reported only after activin stimulation of transfected cells (Lagna et al., 1996). After stimulation with TGF-xcex2 or activin Smad4 interacts with Smad2 or Smad3, and upon BMP challenge a heteromeric complex of Smad4 and Smad1 has been observed (Lagna et al., 1996). Upon ligand stimulation, Smad complexes translocate from the cytoplasm to the nucleus (Hoodless et al., 1996; Liu et al., 1996; Baker and Harland, 1996; Macxc3xadas-Silva et al., 1996), where they, in combination with DNA-binding proteins, may regulate gene transcription (Chen et al., Nature 383:691-696, 1996).
The invention provides isolated Smad7 nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated polypeptides encoded by the Smad7 nucleic acids and agents which bind such polypeptides, including antibodies. The foregoing can be used in the diagnosis or treatment of conditions characterized by the expression of a Smad7 nucleic acid or polypeptide, or lack thereof. The invention also provides methods for identifying pharmacological agents useful in the diagnosis or treatment of such conditions. Here, we present the identification of Smad7, which opposes pathway specific Smads including Smad1, Smad2 and Smad3 and thus is an inhibitor of the TGF-xcex2 superfamily signalling pathways.
According to one aspect of the invention, an isolated nucleic acid molecule is provided. The molecule hybridizes under stringent conditions to a molecule consisting of the nucleic acid sequence of SEQ ID NOs:3 or 5. The isolated nucleic acid molecule codes for a polypeptide which inhibits TGF-xcex2 superfamily signaling. The invention further embraces nucleic acid molecules that differ from the foregoing isolated nucleic acid molecules in codon sequence due to the degeneracy of the genetic code. The invention also embraces complements of the foregoing nucleic acids.
In certain embodiments, the isolated nucleic acid molecule comprises a molecule consisting of the nucleic acid sequence of SEQ ID NO:7 or 8. Preferably, the isolated nucleic acid molecule consists of the nucleic acid sequence of SEQ ID NO:7 or 8, or consists essentially of the nucleic acid sequence of SEQ ID NO:3 or 5.
According to another aspect of the invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule comprises a molecule consisting of a unique fragment of SEQ ID NO:3 between 12 and 1944 nucleotides in length and complements thereof, or a unique fragment of SEQ ID NO:5 between 12 and 1875 nucleotides in length and complements thereof, provided that the isolated nucleic acid molecule excludes sequences consisting only of SEQ ID NO:1 and SEQ ID NO:2. Preferably the isolated nucleic acid molecule excludes molecules consisting solely of nucleotide sequences selected from the group consisting of accession numbers AA061644 (SEQ ID NO:1), AA022262 (SEQ ID NO:2), AA347307, AA348247, 321995, W78627, W40869, AA033426, AA397050, AA016891, and C85115. In one embodiment, the isolated nucleic acid molecule consists of between 12 and 32 contiguous nucleotides of SEQ ID NO:3, SEQ ID NO:5, or complements of such nucleic acid molecules. In preferred embodiments, the unique fragment is at least 14, 15, 16, 17, 18, 20 or 22 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, or complements thereof.
According to another aspect of the invention, the invention involves expression vectors, and host cells transformed or transfected with such expression vectors, comprising the nucleic acid molecules described above.
According to still other aspects of the invention, transgenic non-human animals are provided. The animals include in certain embodiments the foregoing expression vectors. In certain preferred embodiments, the transgenic non-human animal includes a conditional Smad7 expression vector, such as an expression vector that increases expression of Smad7 in a tissue specific, development stage specific, or inducible manner. In other embodiments, the transgenic non-human animal has reduced expression of Smad7 nucleic acid molecules. In some embodiments, the transgenic non-human animal includes a Smad7 gene disrupted by homologous recombination. The disruption can be homozygous or heterozygous. In other embodiments, the transgenic non-human animal includes a conditional Smad7 gene disruption, such as one mediated by e.g. tissue specific, development stage specific, or inducible, expression of a recombinase. In yet other embodiments, the transgenic non-human animal includes a trans-acting negative regulator of Smad7 expression, such as antisense Smad7 nucleic acid molecules, nucleic acid molecules which encode dominant negative Smad7 proteins, Smad7 directed ribozymes, etc.
According to another aspect of the invention, an isolated polypeptide is provided. The isolated polypeptide is encoded by the isolated nucleic acid molecule of any of claims 1, 2, 3, 4, 5 or 6, and the polypeptide inhibits TGF-xcex2 superfamily signaling activity.
In other embodiments, the isolated polypeptide consists of a fragment or variant of the foregoing which retains the activity of the foregoing. In preferred embodiments, the fragment is a C-terminal fragment of Smad7, preferably amino acids 204-426 of SEQ ID NO:4 or SEQ ID NO:6, or a N-terminal fragment of Smad7, preferably amino acids 2-261 of SEQ ID NO:4 or SEQ ID NO:6.
According to another aspect of the invention, there are provided isolated polypeptides which selectively bind a Smad7 protein or fragment thereof, provided that the isolated polypeptide is not a TGF-xcex2 superfamily type I receptor (e.g., a TGF-xcex2, activin or BMP receptor). The isolated polypeptide in certain embodiments binds to a polypeptide encoded by the isolated nucleic acid molecule of any of claims 1, 2, 3, 4, 5 or 6. In preferred embodiments, isolated binding polypeptides include antibodies and fragments of antibodies (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region which binds selectively to an epitope defined by the Smad7 polypeptides of the invention, such as SEQ ID NOs:4 or 6). In other preferred embodiments, the polypeptide is an antibody or fragment thereof which selectively binds an epitope defined by a polypeptide selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In still other preferred embodiments, the isolated polypeptide is a monoclonal antibody, a humanized antibody or a chimeric antibody.
The invention provides in another aspect an isolated complex of polypeptides. The isolated complex includes a TGF-xcex2 superfamily receptor selected from the group consisting of activated TGFxcex2 superfamily type I receptors and complexes of TGFxcex2 superfamily type I receptors and TGFxcex2 superfamily type II receptors (e.g. a TGF-xcex2, activin, Vg1 or BMP receptors) bound to a polypeptide as claimed in claim 1. Preferably the isolated complex includes a polypeptide having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6. In other preferred embodiments, the receptor is selected from the group consisting of Txcex2RI, BMPR-IA, BMPR-IB, ActR-IA, a complex of Txcex2RI and Txcex2RII, a complex of BMPR-IA and BMPR-II a complex of BMPR-IB and BMPR-II, a complex of ActR-IA and BMPR-II and a complex of ActR-IA and ActR-II.
According to still another aspect of the invention, methods for reducing TGF-xcex2 superfamily signal transduction in a mammalian cell are provided. The methods involve contacting a mammalian cell with an amount of an inhibitor of TGF-xcex2 superfamily signal transduction effective to reduce such signal transduction in the mammalian cell. Preferably the TGF-xcex2 superfamily signal transduction is mediated by a TGFxcex2 superfamily ligand, particularly TGF-xcex21, activin, Vg1, BMP-4 and/or BMP-7. Other methods are provided for reducing phosphorylation of pathway specific Smads (e.g. Smad1, Smad2, Smad3 and/or Smad5) by contacting a mammalian cell with the inhibitor disclosed above. In certain embodiments of the foregoing methods, the inhibitor is an isolated Smad7 polypeptide or a fragment thereof, such as a polypeptide encoded by a nucleic acid which hybridizes under stringent conditions to SEQ ID NO:3 or 5, nucleic acids which encode the polypeptide of SEQ ID NO:4 or SEQ ID NO:6 or degenerates or complements thereof. In some embodiments the nucleic acid encodes amino acids 204-426 of SEQ ID NO:4 or SEQ ID NO:6 or amino acids 2-261 of SEQ ID NO:4 or SEQ ID NO:6. In still other embodiments, the inhibitor is an isolated Smad8 polypeptide or a fragment thereof.
According to still another aspect of the invention, methods for modulating proliferation and/or differentiation of a cell are provided. The methods involve contacting a cell with an amount of an isolated Smad7 polypeptide, or a nucleic acid encoding and expressing such a polypeptide, as described above, effective to modulate the proliferation and/or differentiation of the cell.
The invention in a further aspect provides methods for increasing TGF-xcex2 superfamily signal transduction in a mammalian cell. The mammalian cell is contacted with an agent that selectively binds to an isolated nucleic acid molecule of the invention or an expression product thereof in an amount effective to increase TGF-xcex2 superfamily signal transduction. Preferably the TGF-xcex2 superfamily signal transduction is mediated by a TGFxcex2 superfamily ligand selected from the group consisting of TGF-xcex21, activin, Vg1, BMP-4 and BMP-7. Preferred agents are antisense nucleic acids, including modified nucleic acids, and polypeptides including antibodies which bind to the polypeptide including the amino acids of SEQ ID NO:4, the polypeptide including the amino acids of SEQ ID NO:6, the polypeptide including the amino acids of SEQ ID NO:13, the polypeptide including the amino acids of SEQ ID NO:14, a N-terminal fragment of Smad7 or a C-terminal fragment of Smad7, and dominant negative variants of the polypeptide of SEQ ID NO:4 or SEQ ID NO:6.
The invention in still another aspect provides compositions comprising a Smad7 polypeptide and a pharmaceutically acceptable carrier.
The invention in a further aspect involves methods for decreasing Smad7 TGF-xcex2 superfamily inhibitory activity in a subject. An agent that selectively binds to an isolated nucleic acid molecule of the invention or an expression product thereof is administered to a subject in need of such treatment, in an amount effective to decrease TGFxcex2 superfamily signal transduction inhibitory activity of Smad7 in the subject. Preferably the TGFxcex2 superfamily signal transduction is mediated by a TGFxcex2 superfamily ligand selected from the group consisting of TGF-xcex21, activin, Vg1, BMP-4 and BMP-7. Preferred agents are antisense nucleic acids, including modified nucleic acids, and polypeptides including antibodies which bind to the polypeptide including the amino acids of SEQ ID NO:4, the polypeptide including the amino acids of SEQ ID NO:6, the polypeptide including the amino acids of SEQ ID NO:13, the polypeptide including the amino acids of SEQ ID NO:14, a N-terminal fragment of Smad7 or a C-terminal fragment of Smad7, and dominant negative variants of the polypeptide of SEQ ID NO:4 or SEQ ID NO:6.
In another aspect the invention provides methods for diagnosing induction of a TGF-xcex2 superfamily ligand in a cell. The methods include the steps of (a) measuring the amount of Smad7 RNA or polypeptide in the cell and comparing the result of step (a) with a control.
According to still another aspect of the invention, methods are provided for determining the presence of a functional TGFxcex2 superfamily receptor in a cell. The methods include contacting the cell with an amount of TGFxcex2 superfamily ligand effective to increase the amount of Smad7 in the cell, measuring the amount of Smad7 RNA or polypeptide in the cell, and comparing the result of the measurement with a control, wherein an increased amount of Smad7 RNA or polypeptide in the cell indicates the presence of a functional TGFxcex2 superfamily receptor. Preferably the TGFxcex2 superfamily receptor is selected from the group consisting of TGFxcex2 superfamily type I receptors, TGFxcex2 superfamily type II receptors, and complexes of TGFxcex2 superfamily type I receptors and TGFxcex2 superfamily type II receptors.
According to another aspect of the invention, methods are provided for identifying lead compounds for a pharmacological agent useful in the diagnosis or treatment of disease associated with TGFxcex2 superfamily signal transduction inhibitory activity of Smad7. One set of methods involves forming a mixture of a Smad7 polypeptide, a TGF-xcex2 superfamily receptor complex or an activated TGFxcex2 superfamily type I receptor, and a candidate pharmacological agent. The mixture is incubated under conditions which, in the absence of the candidate pharmacological agent, permit a first amount of specific binding of the TGF-xcex2 superfamily receptor complex or activated TGFxcex2 superfamily type I receptor by the Smad7 polypeptide. A test amount of the specific binding of the TGF-xcex2 superfamily receptor complex or activated type I receptor by the Smad7 polypeptide then is detected. Detection of an increase in the foregoing activity in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which increases the TGF-xcex2 superfamily signal transduction inhibitory activity of Smad7. Detection of a decrease in the foregoing activities in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which decreases the TGF-xcex2 superfamily signal transduction inhibitory activity of Smad7. Another set of methods involves forming a mixture as above, adding further a pathway specific Smad polypeptide, and detecting first and test amounts of TGF-xcex2 superfamily induced phosphorylation of the pathway specific Smad polypeptide. Detection of an increase in the phosphorylation in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which decreases the TGF-xcex2 superfamily signal transduction inhibitory activity of Smad7. Detection of a decrease in the foregoing activities in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which increases the TGF-xcex2 superfamily signal transduction inhibitory activity of Smad7. Preferred Smad7 polypeptides include the polypeptides of claim 18.
In the foregoing compositions and methods, preferred members of the TGF-xcex2 superfamily are TGF-xcex21, activin, Vg1, BMP-4 and BMP-7, and the preferred pathway specific Smad polypeptides are Smad1, Smad2, Smad3 and Smad5.
According to yet another aspect of the invention, a method for reducing expression of a Smad6 or Smad7 nucleic acid or expression product thereof in a cell is provided. The method includes contacting the cell with an amount of an agent which binds selectively to Smad4 effective to reduce the expression of the Smad6 or Smad7 nucleic acid or expression product thereof in the cell. In certain embodiments, the agent is an antisense Smad4 molecule, or an antibody that selectively binds to Smad4.
In another aspect, the invention provides a method for increasing Smad6 or Smad7 expression in a cell. The method includes contacting the cell with an agent selected from the group consisting of activin, epidermal growth factor and phorbol esters in an amount effective to increase Smad6 or Smad7 expression in the cell.
According to another aspect of the invention, a method for treating a subject having lung cancer characterized by elevated expression of a Smad6 gene or a Smad7 gene is provided. The method includes administering to the subject an amount of an antisense nucleic acid which binds to the expression product of the Smad 6 or Smad7 gene effective to reduce the expression of the Smad 6 or Smad7 gene. In other embodiments, the method includes administering a polypeptide, such as an antibody or fragment thereof which binds a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:4, a polypeptide comprising the amino acid sequence of SEQ ID NO:6, a N-terminal fragment of Smad7 and a C-terminal fragment of Smad7, a polypeptide comprising the amino acid sequence of SEQ ID NO:10, a polypeptide comprising the amino acid sequence of SEQ ID NO:11, a polypeptide comprising the amino acid sequence of SEQ ID NO:12, a polypeptide comprising the amino acid sequence of SEQ ID NO:13, and a polypeptide comprising the amino acid sequence of SEQ ID NO:14. In still other embodiments, the agent is a dominant negative variant of Smad6 or Smad7.
According to yet another aspect of the invention, methods for reducing eye defects in a developing mammalian embryo are provided. The methods include contacting the cells of the embryo with an agent which reduces the expression or activity of a Smad7 nucleic acid molecule or an expression product thereof. In certain embodiments, the agent selectively binds the Smad7 nucleic acid molecule or an expression product thereof. In preferred embodiments, the agent is an antisense nucleic acid molecule or a polypeptide, and preferably the polypeptide is an antibody or fragment thereof which binds a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:4, a polypeptide comprising the amino acid sequence of SEQ ID NO:6, a polypeptide comprising the amino acid sequence of SEQ ID NO:13, a polypeptide comprising the amino acid sequence of SEQ ID NO:14, a N-terminal fragment of Smad7 and a C-terminal fragment of Smad7. The agent also can be a dominant negative variant of Smad7.
According to another aspect of the invention, an isolated polypeptide is provided. The polypeptide includes a first polypeptide or fragment thereof linked to a second polypeptide or fragment thereof, wherein the second polypeptide or fragment thereof comprises a MH2 domain of Smad7. The polypeptide is localized in the nucleus of a cell and is exported from the nucleus to the cytoplasm of the cell upon TGFxcex2 superfamily receptor-mediated signal transduction in cells having a TGFxcex2 superfamily receptor.
According to another aspect of the invention, a fusion protein is provided which includes a Smad7 MH2 domain or a nuclear localization fragment thereof In other aspects of the invention, a fusion protein is provided which includes a Smad7 MH2 domain or a transcriptional activation fragment thereof.
According to still another aspect of the invention, methods for modulating transcription of Smad7-regulated gene transcription are provided. The methods include contacting a mammalian cell with an agent which modulates TGFxcex2 superfamily receptor-mediated signal transduction in an amount effective to modulate Smad7-regulated gene transcription. In some embodiments the agent is a TGFxcex2 superfamily ligand or an inhibitor of TGFxcex2 superfamily receptor-mediated signal transduction.
In another aspect of the invention, an isolated nucleic acid molecule is provided. The nucleic acid molecule includes the nucleotide sequence of SEQ ID NO:15, an allelic variant thereof, or a functional fragment thereof which confers TGFxcex2 regulation. Nucleic acid molecules which include the nucleotide sequences which hybridize under stringent conditions to SEQ ID NO:15 also are provided. In still other aspects of the invention, isolated nucleic acid molecules which include a unique fragment of SEQ ID NO:15 are provided. Expression vectors which include the foregoing isolated nucleic acid molecules including or related to SEQ ID NO:15 also are provided.
According to another aspect of the invention, method for regulating transcription of a first nucleic acid molecule are provided. The methods include preparing a construct comprising the first nucleic acid molecule operably linked to the foregoing isolated nucleic acid molecules including or related to SEQ ID NO:15, and introducing the construct into an expression system. In some embodiments the expression system is a cell. In preferred embodiments, the cell expresses a TGFxcex2 superfamily receptor, and the method includes contacting the cell with a TGFxcex2 superfamily ligand to increase expression of the first nucleic acid molecule.
In still other aspects of the invention, methods for identifying modulators of TGFxcex2-regulated transcriptional activity are provided. The methods include providing an expression system with a reporter construct including SEQ ID NO:15 or a TGFxcex2-regulated fragment thereof operably linked to a nucleic acid encoding a detectable expression product, and contacting the expression system with a candidate modulator compound. The expression system is incubated under conditions which, in the absence of the candidate modulator, permit a first amount of expression of the detectable expression product. A test amount of the expression the detectable expression product then is detected. Detection of an increase in the foregoing activity in the presence of the candidate modulator compound indicates that the candidate modulator compound is a compound which increases TGFxcex2-regulated transcriptional activity. Detection of a decrease in the foregoing activities in the presence of the candidate modulator compound indicates that the candidate modulator compound is a compound which decreases TGFxcex2-regulated transcriptional activity. In certain preferred embodiments the expression system is a cell or an in vitro transcription system. In other preferred embodiments, the detectable expression product is a reporter protein, such as an enzyme, e.g., luciferase, or a green fluorescent protein.
The use of the foregoing compositions in the preparation of a medicament is also contemplated.
These and other aspects of the invention will be described in further detail in connection with the detailed description of the invention.